SAM 906.06

Page 1 of 11

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 906.06

Supplemental Assay Method for Sterility Testing of Preparations Other Than 

Live Vaccines

Date: May 12, 2023

Number: SAM 906.06

Supersedes: SAM 906.05, July 26, 2018

Standard Requirement: 9 CFR, part 113.26

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 2 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the samples

4. Performance of the Test

5. Examination of the Test Vessels

6. Interpretation of the Test Results

7. Record and Report of Test Results

8. References

9. Summary of Revisions

Appendices
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 3 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

1. Introduction

This Supplemental Assay Method (SAM) describes the test procedure used to detect viable 

bacteria and fungi in all biological products other than live vaccines, per title 9, Code of Federal

Regulations (9 CFR), part 113.26. In the presence of these contaminating extraneous agents, the 

medium will be rendered turbid by macroscopic examination.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 30°- 35°C incubator

2.1.2 20°- 25°C incubator

2.1.3 Laminar-flow Class II biosafety cabinet (BSC)

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Bacillus subtilis (American Type Culture Collection (ATCC) #6633) or 

equivalent organism as specified in the current United States Pharmacopoeia 

(USP)

2.2.2 Issatchenkia orientalis (ATCC #6258) or equivalent organism as 

specified in the current USP

2.2.3 Soybean Casein Digest Medium (SCDM) or Trypticase Soy Broth (TSB)

(National Centers for Animal Health (NCAH) Media #10423) (Appendix I) 

2.2.4 Fluid Thioglycollate Medium (FTM), NCAH Media #10135 (Appendix 

II)

2.2.5 Fluid Thioglycollate Medium with Beef (FTM w/Bf), NCAH Media 

#10227 (Appendix III) 

2.2.6 Trypticase Soy Agar (TSA) plates, NCAH Media #10487 (Appendix IV)

2.2.7 Glassware: tubes and flasks containing test media
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 4 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

2.2.8 Sterile water in serum vials

2.2.9 Lab coat or sterile sleeves and gloves 

2.2.10 70% ethanol

2.2.11 4 x 4-inch sterile gauze pads

2.2.12 Sterile syringes with needles

2.2.13 Vacutainer® needles

2.2.14 Sterile pipettes, individually packaged

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling and disposal of 

biological agents, reagents, tissue culture samples, and chemicals. Personnel must also 

have knowledge of safe operating procedures and policies, as well as training in the 

operation of the necessary laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Turn on the BSC at the beginning of the work week and leave on all week.

3.2.3 Monitor the temperature of incubators, freezers, and coolers according to 

SOPs.

3.3 Preparation of reagents/control procedures

3.3.1 Bacillus subtilis stock culture is prepared according to the manufacturer’s 

instructions and titrated to determine colony forming unit (CFU) concentration.

3.3.2 Issatchenkia orientalis stock culture is prepared according to the 

manufacturer’s instructions and titrated to CFU concentration.
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 5 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

3.3.3 Dilution of Preservative Screening (Eleventh Vessel Positive Control): 

For each serial tested, inoculate an additional container of media for each 

incubation temperature with 1.0 mL of sample and approximately 100 CFU of the 

appropriate indicator organism (Sections 2.2.1 and 2.2.2). This control is used to 

confirm the ratio of inoculum to medium that will result in sufficient dilution of 

the product to prevent bacteriostatic and fungistatic activity according to 9 CFR 

113.25(d).

3.3.4 Maximum Medium Volume Limits per Vessel: The maximum volume of 

media per vessel used will not exceed 500 mL as these large volumes are 

hazardous when removing from the autoclave. Volumes greater than 500 mL will 

be divided evenly into two vessels and the volume of inoculum will be divided 

accordingly.

3.3.5 Negative Control: Include 5 uninoculated test vessels of each media used 

in the testing session to confirm the sterility of the media batch according to 9 

CFR 113.25(c). Incubate the representative test vessels at each incubation 

temperature for 14 days.

3.4 Preparation of the samples

3.4.1 Follow Section V.A of the Outline of Production (OP) for final product(s) 

to determine the type and/or volume of media that should be aliquoted into each 

vessel. The type of media used for testing is based on the product description. 

This information is compiled in Appendix V. Master Cell Stock (MCS), primary 

cells, and ingredients of animal origin are tested with 40 mL of medium at each 

temperature unless otherwise directed. Order a sufficient volume of media to 

accommodate the test vessels, positive controls, negative controls, and extra 

vessels for potential subcultures. 

3.4.2 For products without accompanying diluent, order sterile purified water in 

serum vials in volumes specified on the product label or in the OP.

Note: Most non-live products are liquid; rehydration is only occasionally 

required.

3.4.3 Ten vials of final product and a minimum of 20 mL MCS, primary cells,

and ingredients of animal origin are required for sterility testing.

4. Performance of the Test

4.1 Dress in a clean lab coat or sterile sleeves and gloves to perform sterility testing.
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 6 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

4.2 Wipe down the interior surfaces of the BSC used for testing with 70% ethanol

immediately prior to use.

4.3 Place the necessary testing materials (syringes, Vacutainer® needles, 4 x 4-inch 

gauze squares, etc.), test media, and the product to be tested in the BSC.

4.4 Swab the top of each container of product, diluent, and water with a gauze pad 

soaked in 70% ethanol.

4.5 If necessary, rehydrate each lyophilized sample container of product using a 

syringe and needle or Vacutainer® needle. Use the diluent provided with the product or 

sterile water. For completed product, reconstitute to the minimum volume as indicated on 

the product label or Section VI of the OP.

4.6 Withdraw product from each container with a new sterile syringe and needle. 

Dispense a 1.0 mL aliquot from each product vial into one vessel of each media type used 

for testing (one vessel per incubation temperature). If the recoverable volume from an 

individual container of product is less than 2.0 mL, inoculate 1⁄2 the sample volume into 

each test vessel. For MCS, primary cells, and ingredients of animal origin, dispense 1.0 

mL aliquots into each test vessel. Swirl each test vessel after inoculating with sample to 

distribute the product in the media.

4.7 Inoculate one additional vessel of each type of media with 1.0 mL of product to

serve as the 11th vessel positive controls (see Section 3.3.3). The product sample for these 

control vessels may be obtained from any of the previously used containers or from an

eleventh vial of product. Set the 11th vessels to the side and continue with the testing 

session.

Note: The 11th test vessel is not conducted with MCS, primary cells, and ingredients

of animal origin. 

4.8 When all of the test vessels have been inoculated with product, set-up the negative 

control for the testing session (see Section 3.3.5). 

4.9 Once the sterility portion of the test has been completed, prepare the positive 

control organisms in an area that is separate and apart from the clean area where the 

sterility test was conducted (see Sections 3.3.1 and 3.3.2). The type of organism to be 

used for each product is specified in Appendix V.

4.9.1 Inoculate approximately 100 CFU of the appropriate indicator organism 

into the vessels prepared in Section 4.7 and swirl the vessel to distribute the 

organism in the medium. 

4.9.2 After the 11th test vessels have been inoculated with the indicator 

organisms, inoculate one TSA plate per indicator organism with a representative 
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 7 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

volume of inoculum. This plate count serves to demonstrate that the appropriate 

number of viable organisms were added to the test vessel.

4.10 Incubate the test vessels and the representative control at the appropriate 

incubating temperature. Incubate all vessels for 14 days. Incubate the TSA plates 

containing the indicator organisms for a maximum of 7 days.

4.11 Wipe down the interior of the BSC and counter tops with 70% ethanol at the end 

of the testing session. Discard biological samples and contaminated materials according 

to SOPs.

5. Examination of the Test Vessels

5.1 Examine all test vessels for cloudiness or turbidity at least once during the testing 

period. Observe bacterins, bacterin-toxoids and clostridial toxoids on days 7 to 11; MCS, 

primary cells, and ingredients of animal origin on days 3-7; and observe all samples on 

test on day 14.

5.2 Determine if the cause of cloudiness or turbidity is due to microbial growth by 

subculturing the contents of the test vessel by aseptically transferring 1.0 mL from the 

test vessel in question to 20 - 25 mL of fresh test media. Swirl the vessel to mix the 

inoculum into the media and incubate at the appropriate temperature for a minimum of 3 

days. In addition, subculture the test vessel culture onto two plates of blood agar and TSA 

and streak for isolation. Incubate one plate of each at both incubation temperatures for 3 

days. 

5.3 On day 14, vessels without growth are considered negative. 

5.4 By day 14, any vessels with bacterial or fungal growth confirmed by growth in 

the subcultures are considered positive for extraneous growth. The contaminant will also 

be identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass 

Spectrometry (MALDI-TOF MS).

6. Interpretation of the Test Results

6.1 Criteria for a valid test:

6.1.1 There must be no growth in the Negative Control vessels.

6.1.2 For final products tested for dilution of preservative at the Center for 

Veterinary Biologics, the TSA plates containing B. subtilis must contain an 

average count of 93-128 CFU and the TSA plates containing I. orientalis must 

contain an average count of 76-124 CFU.
Center for Veterinary Biologics SAM 906.06 

Testing Protocol Page 8 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

Note: A range of approximately 100 CFU should be determined at each biologics 

manufacturer facility for each new positive control lot. 

6.1.3 For final products tested for dilution of preservative, growth of the 

indicator organism must be observed in the 11th vessel positive control. 

6.1.4 If these criteria are not met, the test is considered invalid or a no test (NT). 

Products may be reported and released with a NT result if there is no reason to 

suspect an unsatisfactory sterility result of that product. 

6.2 If no growth is found in any test vessel, the serial is satisfactory (SAT). 

6.3 If growth is observed in any test vessel and confirmed by subculture, the test may 

be repeated using 20 unopened final container samples. 

6.4 If extraneous growth is found in any test vessel of the final test, the serial is 

unsatisfactory (UNSAT). 

6.5 If there is no growth in the 11th vessel positive control(s), there will be a 3-week 

delay before the test results are reported (Center for Veterinary Biologics Notice 09-02,

http://www.aphis.usda.gov/animal_health/vet_biologics/publications/notice_09_02.pdf) 

while a dilution of preservative study will be conducted on the serial according to 9 CFR 

113.25(d). If the volume of media listed in the OP for sterility testing of the serial 

indicates interference in the 9 CFR 113.25(d) test, this testing will be reported as UNSAT 

and the sterility test result will be reported as a NT. 

7. Record and Report of Test Results

Record and report results of the test(s) according to SOPs. 

8. References

8.1 Title 9, Code of Federal Regulations, parts 113.25 and 113.26, U.S. Government 

Printing Office, Washington, DC. 

8.2 The U.S. Pharmacopoeia, 1985, Vol. 21, pp. 1151-1160, Mack Publishing Co., 

Easton, PA. 

8.3 Kurtzman, C. P., C. J. Robnett, and E. Basehoar-Powers. 2008. Phylogenetic 

relationships among species of Pichia, Issatchenkia and Williopsis determined from 

multigene sequence analysis, and the proposal of Barnettozyma genera novel, Lindnera 

genera novel and Wickerhamomyces genera novel. FEMS Yeast Res 8:939-54. 
Center for Veterinary Biologics SAM 906.06 

Testing Protocol Page 9 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

9. Summary of Revisions 

Version .06 

• Updated cover page.

• Adjusted Bacillus subtilis acceptable validity range.

Version .05 

• Updated cover page.

• Updated Sections 2 through 6.

Version .04 

• The Bacteriology Section Leader has been updated. 

• Updated Sections 2.2, 3.3, and 4. 

• Deleted Appendix V (Blood Agar base with 5% bovine blood – NVSL Media 

#10006). 

Version .03

• Revised to include dilution of preservative requirements cited in CVB Notice 09-02. 

• The Contact information has been updated. 

• Sections 3.3, 4, 5, and 6 have been updated to reflect current practices.

• 8.3: Reference added for name change of Candida krusei to Issatchenkia orientalis. 

• Appendices: Updated media storage limits to be in compliance with 9 CFR 

113.25(b). 

Version .02 

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail and the following changes were made to the document: 

• The Contact has been changed from Gerald Christianson to Sophia Campbell and 

Janet Wilson.

• 2.1 The Bunsen burner has been removed from the list of equipment that is needed 

for the test.

• 3.3.3: This section has been added to describe the 11th vessel positive control. 

• 3.3.4: This section has been added to clarifiy media vessel volume limits used in 

testing. A note on the safety hazard when removing volumes > 500 mL from the 

autoclave has also been added.

• 3.4.3 The practice of looking up the volume of test media needed for each serial to be 

tested on the dilution of preservative computer disk using the notebook program has 

been removed.

• 6.3: Clarification on how to interpret the 11th vessel positive control results has been 

added, along with information on follow-up testing. 

• Brain heart infusion agar and Trypticase soy agar with 5% sheep’s blood have been 

removed from this outline and replaced with blood agar base with 5% bovine blood 

plates and trypticase soy agar plates. 
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 10 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

• Media storage information has been added in the Appendices.
Center for Veterinary Biologics SAM 906.06

Testing Protocol Page 11 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

Appendix I

Trypticase Soy Broth (TSB) or Soybean Casein Digest Medium (SCDM) - NCAH Media 

#10423 

Trypticase Soy Broth 30 g

QH2O 1000 mL

Autoclave 20 minutes at ≥121°C. Store at 20°- 25°C for up to 90 days.

TSB and SCDM are synonymous mediums.

Appendix II

Fluid Thioglycollate Medium (BBL) – NVSL Media #10135 

Fluid Thioglycollate Medium 29.5 g

QH2O 1000 mL

Mix and heat to boiling. Autoclave 20 minutes at ≥121°C. Store at 20°- 25°C for 

up to 90 days.

Appendix III

Fluid Thioglycollate with Beef Extract – NVSL Media #10227 

Fluid Thioglycollate Medium 29.5 g

QH2O 1000 mL

Heat and add:

0.5% Beef Extract (DIFCO) 5 g

Bring to a boil and dispense. Autoclave 20 minutes at ≥121°C. Store at 20°- 25°C 

for up to 90 days.

Appendix IV

Trypticase Soy Agar (TSA) – NVSL Media #10487

Trypticase Soy Agar 40 g

QH2O 1000 mL

Boil to dissolve and autoclave for 15 minutes at ≥121°C. Store at 2°- 7°C for up 

to 90 days.
Center for Veterinary Biologics SAM 906.06 

Testing Protocol Page 12 of 12

Supplemental Assay Method for Sterility Testing of Preparations Other Than Live Vaccines

Appendix V 

Media, incubation conditions, and control organisms to be used with each class of 

biological product

Product Description Media/Incubation temp. Control Organism

Killed products without 

merthiolate or clostridial 

components

FTM 30°-35°C Bacillus subtilis

SCDM 20°-25°C Issatchenkia orientalis

Killed products with merthiolate

but no clostridial components 

FTM 30°-35°C Bacillus subtilis

FTM 20°-25°C Issatchenkia orientalis

Killed products with clostridial 

components but no merthiolate 

FTM w/Bf 30°-35°C Clostridium chauvoei*

and

Bacillus subtilis**

SCDM 20°-25°C Issatchenkia orientalis

Killed products with clostridial 

components and merthiolate 

FTM w/Bf 30°-35°C Clostridium chauvoei* 

and

Bacillus subtilis**

FTM 20°-25°C Issatchenkia orientalis

Plasma bags without merthiolate

FTM 30°-35°C Bacillus subtilis

SCDM 20°-25°C Issatchenkia orientalis

Plasma bags with merthiolate

FTM 30°-35°C Bacillus subtilis

FTM 20°-25°C Issatchenkia orientalis

Program disease products without 

merthiolate 

FTM 30°-35°C Bacillus subtilis

SCDM 20°-25°C Issatchenkia orientalis

Program disease products with 

merthiolate 

FTM 30°-35°C Bacillus subtilis

FTM 20°-25°C Issatchenkia orientalis

 *9 CFR 113.25(b) testing. 

 **11th Vessel testing.